(a) Field of the Invention
This invention relates, in general, to a method for detecting the presence of antigens associated with hepatitis. In particular, it relates to an immunoassay for antigens associated with hepatitis involving the use of an enzyme-tagged antibody which reacts with an antigen to detect the presence of hepatitis.
(b) Description of the Prior Art
Hepatitis, which means "an inflammation of the liver", is due to an infection or obstruction of the bile channels. There are thought to be two varieties of viral hepatitis, one having a longer incubation period than the other. In the past, when a patient contracted hepatitis and had a known parenteral exposure, the hepatitis was termed "serum hepatitis". If the patient did not have a known parenteral exposure and he contracted hepatitis orally it was called "infectious". However, it has been documented that in addition to having overlapping incubation periods, "infectious hepatitis" can be contracted parenterally, and, the so-called "serum hepatitis" can be contracted orally. Thus, although there would appear to be two forms of hepatitis caused by at least two distinct agents, the terms "serum hepatitis" and "infectious hepatitis" should not be used to distinguish them. Accordingly, it has been suggested that the terms "hepatitis A" be used to designate the form most closely resembling "infectious hepatitis", and, the term "hepatitis B" be used to designate the form most closely resembling "serum hepatitis".
The examples appearing in this specification are directed to the detection of the antigen or antigens associated with hepatitis type B. Patients who contract the form most closely resembling serum hepatitis, no matter how contracted, often have these antigens in their blood. At this point, it should be noted that there is no reliable assay for determining the presence of an antigen associated with hepatitis A or an hypothesized hepatitis C. Thus, the examples in this specification are directed to the detection of the presence of those antigens associated with hepatitis B. However, there is no reason why the process of the present invention could not be used to detect the presence of antigens associated with other types of hepatitis once their antigens have been identified.
Contraction of "serum hepatitis" or hepatitis B creates a serious clinical problem that cannot be ignored. Because of the severity of this problem, a variety of test methods for the detection of hepatitis have been developed. These included Micro-Ouchterlony, immunodiffusion, complement fixation, immunoelectro-osmophoresis, haemagglutination and haemagglutination inhibition, electron microscopy, and solid phase radioimmunoassay. See British Medical Bulletin, 1972, Vol. 28, No. 2 (Viral Hepatitis) pages 138-141 for a brief description of each.
Immunoelectro-osmophoresis or counterelectrophoresis (CEP) provides a rapid, simple method for the detection of the hepatitis antigen and its antibody. However, this technique is somewhat less sensitive than, for example, complement fixation. Its principle advantage is that tests can be completed within two hours. However, because of its low sensitivity level CEP is no longer approved by the Food and Drug Administration.
The application of radio-immunoassay (RIA) for routine diagnostic purposes is believed to be somewhat limited, not only because of the relatively complex, specialized, and expensive equipment necessary for conducting the test, but also because of the strict precautions required in handling radio-active isotopes. Isotope tagging presents a serious potential health hazard, requires monitoring and Atomic Energy Commission licensing (for user and manufacturer), and presents waste disposal problems. Nevertheless, this technique is now rather well established for immunoassay.
Immunological methods depend, of course, upon a primary characteristic of all antibodies and antigens, i.e., their ability to react with a specific complimentary antigen or antibody. Thus, if an antibody is added to a serum containing its antigen, the antibody and antigen will complex and may precipitate from the solution. In most of the above-mentioned test methods, the presence of antigens in human sera is detected by making use of this simple fact.
Labeled antibodies have been used previously for identifying various antigens. If an antibody known to be specific for a particular antigen is isolated from the globulin portion of serum or plasma of a host animal which has been stimulated to produce that antibody, it can be labeled or tagged by known means. By conjugating the antibody with a labeling agent, e.g., a physically detectable substance such as a radio isotope, as above-mentioned, or fluorescent chemicals, the presence of the antibody can be detected. Thus, when used diagnostically, if the counterpart antigen is present in some prepared test sample, the labeled antibody will attach itself to that antigen, and the presence of the antigen can be confirmed through detection of the labeled antibody in the sample.
A labeled antibody, for diagnostic purposes, should be made sufficiently specific so that it will react only with those antigens whose detection is desired and without cross-reaction with other closely related antigens which may have quite dissimilar or insignificant consequences. Thus, it is apparent that both the source and the manner of preparation of the antibody is quite critical in any immunoassay.
One manner of detecting hepatitis associated antigen, as earlier mentioned, involves solid phase radio-immunoassay. Such a procedure is disclosed in U.S. Pat. No. 3,867,517. As disclosed therein, in the performance of the assay, a tube well, or insert for use therewith, of molded polystyrene is first coated with antibody. This is accomplished by incubating the member to be coated with an antibody solution. Afterwards, the unknown sample is incubated with the coated well or insert to react the antibody with antigen present in the sample. The well is then washed and incubated with an antibody labeled with the radioactive isotope I-125. It is then again washed to remove any unbonded labeled antibody. Thus, in the event any antigen is present in the test sample, a sandwich is formed from the polystyrene well (or insert), the antibody, the antigen, and the I-125 tagged antibody. The radiation emitted from the I-125 tagged antibody is then counted and compared against a control.
It has also been disclosed that a disc of polytetrofluorethylene onto which is grafted a substituted polystyrene, e.g., isothiocyanostyrene, might be useful in performing radioimmunoassay. This polystyrene is an insoluble material having specifically designed surfaces of protein-reactive groups which may be used to covalently bond proteins to provide a reagent useful in bioassay procedures.
Recently, an important alternative to labeling antibodies with radio-isotopes or fluorescent chemicals has been developed. This involves labeling or tagging an antibody with an enzyme. Such a procedure is described in U.S. Pat. Nos. 3,654,090 and 3,791,932. In Clinica Chemica Acta, 48 (1973) 15-18, an enzyme-linked immunoassay for alphafetoprotein by either the competitive or the sandwich procedure is disclosed.
Compared to radio-immunoassay, enzyme labeling offers several important advantages. For example, every enzyme tagged molecule in the final mixture participates in the readout. On the other hand, only a very small proportion of isotopic atoms in the final mixture undergo decay during readout to participate in the assay. In enzyme tagging, every tag repeatedly participates in readout, by attacking many substrate molecules to form a detectable end product, e.g., up to 100,000 times per minute, hence greatly enhancing sensitivity. An isotope tagged atom decays only once during readout, after which it is lost from participation. An enzyme tagged reagent has a long shelf life; whereas, the isotope tagged reagent is constantly decaying, and presents serious shelf life problems. While there is minimal health hazards associated with using an enzyme tagged reagent, serious potential health hazards are encountered with isotope tagging. Lastly, an immunoassay involving enzyme tagging can use simple, relatively inexpensive equipment for readout. The success of isotope immunoassay, in contrast, is dependent on the efficiency of detecting decay, and hence on the quality of very expensive detection equipment.
Although the finding of an antigen associated with hepatitis in one's blood may not be the equivalent of obtaining a clinical history of hepatitis, investigations have revealed a high incidence of hepatitis infection when a patient has received blood which tests positively for antigens associated with hepatitis. Since decisions on whether to use particular blood units available from a blood center must often be made in a relatively short time period, a sensitive, rapid, easy to perform screening test for hepatitis, without need for expensive equipment, is of extreme importance. Although the various tests used in the past for the detection of the antigens associated with hepatitis have been satisfactory to a degree, they are all attendant with one or more disadvantages.